HMOX1 Is a Targeted Therapy for Myeloma-Induced Bone Disease.

Abstract 2944

Multiple myeloma (MM) bone disease is characterized by increased activity of osteoclasts and reduced osteoblast numbers. We recently reported that cytotherapy with mesenchymal stem cells (MSCs) promotes bone formation, inhibits bone disease and reduces MM growth (Yaccoby et al., 2006; Li et al., 2011) and that MSCs secrete bone remodeling associated factors such as decorin that directly inhibits osteoclastogenesis and promotes osteoblastogenesis (Li et al., 2012). To shed light on molecular mechanisms associated with the cytotherapeutic effects of MSCs we exploited the SCID-hu model engrafted with MM cells from various patients and cytotherapeutically treated with fetal or healthy donors MSCs. Human global gene expression profile was performed in nonmyelomatous implanted bones (n=5) and in myelomatous implanted bones injected with human MSCs (1×10⁶ cells/bone) and analyzed immediately (control group, n=17) or 24 hours later (n=16). We indentified heme oxygenase 1 (HMOX1) as one of the top significant upregulated genes consistently induced by 24 hours cytotherapy using fetal MSCs or MSCs from 3 different donors. HMOX1 induction was confirmed by qRT-PCR and immunohistochemistry and was also found to be consistently induced by MSCs cytotherapy in myelomatous bones engrafted with MM cells from different patients (n=4). Further analysis revealed lower expression of HMOX1 in myelomatous versus nonmyelomatous bones. HMOX1, an inducible antioxidant, degrades intracellular heme into carbon monoxide, free iron and biliverdin and is involved in oxidative stress response and intracellular iron homeostasis both of which are known to regulate osteoclastogenesis. Culture of human blood mononucleated cells or committed osteoclast precursors with MSCs (non-contact co-culture) in osteoclastogenic medium supplemented with RANKL and M-CSF inhibited multinucleated osteoclast formation by 70% (p<0.0001) and 97% (p<0.0001), respectively. MSCs induced HMOX1 expression in osteoclast precursors by 3 folds (p<0.02) and HMOX1 levels remained higher in these cells during osteoclast formation. Concurrently, RANK (RANKL receptor) and NFATC1 (osteoclast main transcription factor) were constantly downregulated in osteoclast precursors by MSCs by 65±10% (p<0.01) and 42±11% (p<0.05), respectively, followed by reduced expression of the osteoclastic markers cathepsin K (p<0.0002), acid phosphatase 5 (p<0.01) and vitronectin receptor (p<0.02) at the end of the differentiation process (7 days). Similar to MSCs, overexpression of HMOX1 in osteoclast precursors using lentiviral vector markedly reduced their ability to form osteoclasts while HMOX1 inducer, hemin (50 μM), induced HMOX1 in osteoclast precursors and inhibited their differentiation into osteoclasts by 49±2% (p<0.0001). In MM-bearing SCID-rab mice (9 mice/group) bone mineral density (BMD) was reduced by 17±3% from pretreatment levels in control group whereas in hosts treated with hemin for 3 weeks BMD was reduced by 2±3% (p<0.005). Histological bone sections revealed reduced number of TRAP-expressing osteoclasts in myelomatous bones from hemin-treated hosts (p<0.01). These data suggest that HMOX1 is suppressed in myelomatous bone and that therapeutic induction of HMOX1 is a promising approach to control MM-induced osteoclastogenesis and osteolytic bone disease.